Effects of Column and Mobile Phase Polarity using Steroids as Probes in Packed-Column Supercritical Fluid Chromatography

Abstract

Decreasing stationary phase polarity is ineffective in improving the separation of keto and hydroxy steroids by packed-column supercritical fluid chromatography. Only polar modifiers used with polar stationary phases produce both good resolution and symmetrical peaks. The identity of the bonded stationary phase determines retention but only slightly changes selectivity. Polar stationary phases used with low polarity modifiers, low polarity stationary phases used with pure carbon dioxide, modified carbon dioxide (low or high polarity modifiers), or even modified carbon dioxide containing very polar additives all produce poor peak shapes. On polar stationary phases methanol concentrations above 1–2% produce symmetrical peaks and log k' is a linear function of mobile phase solvent strength. Thus, the separation mechanism involves a single fluid-fluid partition mechanism. Below 1–2% methanol, both adsorption and partition mechanisms operate simultaneously, sometimes producing severely asymmetric peaks. Both keto and hydroxy steroids are separated through normal phase mechanisms.